The amino acid sequence of a 58-residue segment of the small peptide released by chymotrypsin from the COOH-end of the heavy chains of Acanthamoeba myosin II has been determined. The first 36-residues have a sequence predicting an Alpha-helical coiled coil conformation. The three regulatory phosphorylatable serines are at positions 46, 51, and 56 in repeating pentapeptides of nearly identifical sequence. The myosin II molecule missing this small peptide is unable to form bipolar filaments although it does form parallel dimers. It has full Ca++ ATPase activity but no actin-activated ATPase activity nor does it bind to F-actin in the presence of ATP. These and other data support the idea that only filaments of myosin II with the proper conformation (dephosphorylated) have actin-activated ATPase activity. The catalytic and regulatory domains of Acanthamoeba myosins IA and IB have been mapped. They are near each other in the NH2-terminal halves of the heavy chains with the catalytic sites being closer to the terminus. The actin-activated ATPase activity of myosins IA and IB show an actin-dependent myosin cooperatively which may explain the complex enzyme kinetics reported previsouly. Single-headed myosin I is able to crosslink actin filaments suggesting that the myosin either have two actin-binding sites or, in the presence of F-act, can form oligomers. Beads coated with myosin I show ATP-dependent movement along actin cables demonstrating that these single-headed enzymes are able to support motile activity.